Sensation-improving agent

ABSTRACT

An object of the present invention is to provide a safety sensation-improving agent that can improve dulled peripheral sensations through daily ingestion or application to the skin. Another object of the present invention is to provide a sensation-improving food, beverage, feed, or cosmetics that can improve dulled peripheral sensations through oral ingestion or application to the skin. A sensation-improving agent containing a milk-derived protein and/or a hydrolysate therefrom as an active ingredient is provided. The milk-derived protein and/or the hydrolysate therefrom can be orally ingested or applied direct to the skin to improve dulled sensations, particularly peripheral sensations, and be formed into a sensation-improving food, beverage, feed, or cosmetics.

TECHNICAL FIELD

The present invention relates to a sensation-improving agent thatcontains a milk-derived protein and/or a hydrolysate therefrom as anactive ingredient, the sensation-improving agent has an effect ofimproving dulling of peripheral nerves. And the present inventionrelates to a sensation-improving food, beverage, feed, or cosmetics thatincludes the sensation-improving agent.

BACKGROUND ART

In recent years, increases in age-related diseases such as osteoporosisand dementia have become a serious social issue associated with aging.Various drugs have been developed to prevent or cure these age-relateddiseases. However, side effects of such drugs always need to be takeninto consideration. Recently, attempts have been made to prevent or cureage-related diseases through a reconsideration of dietary habits oringestion of a specific food ingredient. For example, ingestion of abasic protein in bovine milk is known to prevent or cure osteoporosis.Furthermore, a preventive and therapeutic agent against Alzheimer'sdefects of memory, containing sphingomyelin, a relatively abundantphospholipid in bovine milk, as an active ingredient is known.

An example of the age-related symptoms includes dulling of peripheralsensations, which is caused by not only aging, but also diseases such asdiabetes. The dulling of peripheral sensations may lead to troubles, forexample, a higher risk of suffering burns caused by failure to feel hotrightly on touching a hot object, or a risk of delaying the discovery ofan injury caused by a dull sensation of pain. In recent years, studiesthat reduce dulling of peripheral sensations caused by aging or diseaseshave been conducted in order to prevent such risks. For example, it hasbeen reported that exogenous ceramide and the enzymes sphingomyelinaseand phosphatidylcholine-specific phospholipase C, the enzymes increasebiosynthesis of endogenous ceramide, promote morphologicaldifferentiation of P-12 cells being neural model cells through 3T3 cellsbeing an established fibroblast cell line, that is, a neurotrophicfactor secreted by 3T3 cells (Non Patent Document 1). The promotion ofmorphological differentiation of the neural model cells indicates theeffect of improving dulling of peripheral sensations. However, use ofthe above ceramide and enzymes, which are not food ingredients, requiresexamination of their safety. In such situations, there is a need for asafer agent that can improve dulled peripheral sensations through dailyingestion or application to the skin.

Components in milk are known to have many physiological activities. Forexample, milk-derived sphingomyelin and phospholipids are known to havean effect of improving dulling of peripheral sensations (Patent Document1 and Patent Document 2). However, the effect of milk-derived proteinson improvements in dulling of peripheral sensations is not yet known.Examples of the milk-derived proteins include lactoperoxidase,lactoferrin, cystatin, and angiogenin.

Lactoperoxidase, present in milk, is a heme iron-containingglycoprotein, while details on its structure are yet to be known.Lactoperoxidase has been found to have an effect of inhibiting in vivoproduction of lipid peroxides and is used as an anti-aging agent thatprevents loss of sight and motor skills, and decline in immune functionsand the like, and a liver function improvement agent. Also, theglycoprotein is known to be used as a low-cariogenic nutrientcomposition due to its low cariogenicity. However, it is yet to be knownthat lactoperoxidase and an enzymatic hydrolysate therefrom producedwith a protease have an effect of improving dulling of peripheralnerves, and are of use as a sensation-improving agent.

Lactoferrin and its hydrolysate are known to have an effect ofpreventing adhesion of pathogens to cells and an antiviral action.Moreover, the mixture with an epidermal growth factor is reported toincrease a skin cell activating effect of the epidermal growth factoralone. Also, lactoferrin is generally known to have an effect ofalleviating stress associated with pain and emotional stress. However,it is yet to be known that lactoferrin and an enzymatic hydrolysatetherefrom produced with a protease have an effect of improving dullingof peripheral nerves, and are of use as a sensation-improving agent.

Cystatin is a cysteine protease inhibitor that inhibits the proteolyticactivity of a cysteine protease having a SH group in the active center,and is found in animal tissues, cells, blood and urine. Also, cystatin'seffect of inhibiting virus growth is found to be a beneficial effect.However, it is yet to be known that cystatin and an enzymatichydrolysate therefrom produced with a protease have an effect ofreducing dulling of peripheral nerves, and are of use as asensation-improving agent.

Angiogenin is one of angiogenesis factors. Human angiogenin is known tobe a protein having a molecular weight of 14,400, and is present inblood and milk. Bovine angiogenin is isolated from bovine milk and issubjected to amino acid sequencing, and the results have already beenreported. A production of angiogenin from bovine milk by subjecting milkto cation exchange chromatography to apply the milk on the cationexchange column, eluting the adsorbate with an alkali metal saltsolution of a weak organic acid, and subjecting the resulting eluate tocation exchange chromatography again and gel filtration chromatographyto collect angiogenin is disclosed. Moreover, angiogenin is found tospecifically inhibit melanin production in melanoma B-16 cells, and isreported to be used also as a possible whitening agent. However, it isyet to be known that angiogenin and an enzymatic hydrolysate therefromproduced with a protease have an effect of improving dulling ofperipheral nerves, and are of use as a sensation-improving agent.

RELATED ART DOCUMENT Patent Document

-   Patent Document 1: Japanese Patent Application Laid-Open Publication    No. 2009-126787-   Patent Document 2: Japanese Patent Application Laid-Open Publication    No. 2009-126788

Non Patent Document

-   Non Patent Document 1: Annual Report of Cosmetology, vol. 10, (2002)

Disclosure of The Invention Problems to be Solved by the InventionSUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a safetysensation-improving agent that can improve dulled peripheral sensationsthrough daily ingestion or application to the skin. Another object ofthe present invention is to provide a sensation-improving food,beverage, feed, or cosmetics that can improve dulled peripheralsensations through oral ingestion or application to the skin.

Means for Solving the Problems

The present inventors, who have diligently pursued a safe componenthighly effective for the improvement of dulled sensations in view ofthese problems, found that oral ingestion or direct application to theskin of any milk-derived protein and/or any hydrolysate therefrom canimprove the dulling of sensations, particularly peripheral sensations.Use of such a milk-derived protein and/or a hydrolysate therefrom as anactive ingredient has completed a sensation-improving agent. The presentinventors also found that the sensation-improving agent can be addedwith a food, beverage or a feed to form a sensation-improving food,beverage, feed, or cosmetics, and have completed the present invention.Throughout the specification, lactoperoxidase, lactoferrin, cystatin,and angiogenin, which are present in milk, are referred to as“milk-derived proteins”. These proteins in use for thesensation-improving agent according to the present invention may notnecessarily be derived from milk. For example, they may be synthesizedartificially or be purified from blood.

The present invention relates to the following aspects:

(1) A sensation-improving agent containing a milk-derived protein and/ora hydrolysate therefrom as an active ingredient;(2) The sensation-improving agent according to Aspect (1), wherein themilk-derived protein is at least one selected from lactoperoxidase,lactoferrin, cystatin, and angiogenin;(3) The sensation-improving agent according to Aspect (1) or (2),wherein the hydrolysate from the milk-derived protein is producedthrough hydrolisis of the milk-derived protein with a protease;(4) The sensation-improving agent according to Aspect (3), wherein theprotease is at least one selected from the group consisting of pepsin,trypsin, chymotrypsin, and pancreatin;(5) A sensation-improving food, beverage, feed, or cosmetics containingthe component according to any one of Aspects (1) to (4);(6) A method for improving a sensation in a mammal, including taking themammal a milk-derived protein and/or a hydrolysate therefrom, orapplying the milk-derived protein and/or the hydrolysate therefrom tothe skin of the mammal; and(7) The method according to Aspect (6), wherein the mammal is a human,and the milk-derived protein and/or the hydrolysate therefrom is fed ata dose of 10 mg or more per day for an adult human.

Effect of Invention

The sensation-improving agent according to the present invention canprovide an effect of improving dulling of peripheral sensations.

DESCRIPTION OF EMBODIMENTS

The present invention is characterized by a milk-derived protein and/ora hydrolysate therefrom as an active ingredient. Examples of themilk-derived protein include lactoperoxidase, lactoferrin, cystatin, andangiogenin. These milk-derived proteins may be prepared from milk ofmammals such as human, cattle, buffalo, goat, and sheep, or be producedby a genetic engineering procedure. Hydrolysates of these milk-derivedproteins can be prepared from the milk-derived proteins by the action ofa protease.

Lactoperoxidase is prepared from milk of mammals. The examples of thesource of milk include mammals such as human, cattle, buffalo, goat, andsheep. Lactoperoxidase, which is a known and commercially availablesubstance, can be industrially produced using known methods, forexample, a method for purifying lactoperoxidase with a sulfonatedcarrier (Japanese Patent Application Laid-Open Publication No.3-109400). Lactoperoxidase produced by a genetic engineering procedurecan also be used in the present invention. A hydrolysate fromlactoperoxidase is a peptide mixture prepared by limited proteolysis ofthe above-described lactoperoxidase with a protease such as trypsin,pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin,thermolysin, or V8 protease so as to have a molecular weight of 10,000or less. The lower limit of the molecular weight is preferably 500 ormore.

Lactoferrin is prepared from milk of mammals. The examples of the sourceof milk include mammals such as human, cattle, buffalo, goat, and sheep.Lactoferrine, which is a known and commercially available substance, canbe industrially produced using known methods, for example, a method forpurifying lactoferrin with a sulfonated carrier (Japanese PatentApplication Laid-Open Publication No. 3-109400). Lactoferrin produced bya genetic engineering procedure can also be used in the presentinvention. A hydrolysate from lactoferrin is a peptide mixture preparedby limited proteolysis of the above-described lactoferrin with aprotease such as trypsin, pancreatin, chymotrypsin, pepsin, papain,kallikrein, cathepsin, thermolysin, or V8 protease so as to have amolecular weight of 10,000 or less. The lower limit of the molecularweight is preferably 500 or more.

Cystatin from any source can be used, including one derived from milk ofmammals such as human, cattle, buffalo, goat, and sheep. For example,the gene sequence of cystatin derived from human milk and bovine milkhas already been determined; hence cystatin can be produced byrecombinant production, and cystatin produced by a genetic engineeringprocedure can also be used in the present invention. Alternatively,cystatin, which is relatively abundant in bovine colostrum, may becollected from the milk. Cystatin can also be collected from a cellculture medium, and such cell culture medium-derived cystatin may beused. For example, milk-derived cystatin can be produced in accordancewith a known method (Japanese Patent Application Laid-Open PublicationNo. 2000-281587) from milk such as raw milk, milk powder, skim milk, andreconstituted milk through treatments such as heat treatment, saltingtreatment, ethanol treatment, various chromatographic treatments such asion exchange chromatography and gel filtration chromatography, andultrafiltration treatment. A hydrolysate from cystatin can be a peptidemixture prepared by limited proteolysis of the cystatin with a proteasesuch as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein,cathepsin, thermolysin, or V8 protease so as to have a molecular weightof 8,000 or less. The lower limit of the molecular weight is preferably500 or more.

For sources of angiogenin, colostrum within 1 to 7 days afterparturition, particularly preferably within 1 to 5 days afterparturition obtained from mammals such as human, cattle, buffalo, goat,and sheep is suitable because such colostrum has a high angiogenincontent, although milk during the original lactation period can also beused as a raw material in the present invention. Angiogenin can beindustrially produced using known methods, for example, a method forpurifying angiogenin by a combination of cation exchange chromatographyand gel filtration chromatography (Japanese Patent Application Laid-OpenPublication No. 2-296000). A hydrolysate from angiogenin is a peptidemixture prepared by limited proteolysis of the above-describedangiogenin with proteases such as trypsin, pancreatin, chymotrypsin,pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease so asto have a molecular weight of 10,000 or less. The lower limit of themolecular weight is preferably 500 or more.

The sensation-improving agent according to the present invention may beused as the above-described milk-derived protein, particularlylactoperoxidase, lactoferrin, cystatin, and angiogenin, or theirhydrolysates prepared from the milk-derived protein by the action of aprotease, may be mixed with other raw materials, such as saccharides,lipids, proteins, vitamins, minerals, and flavors, commonly used forpharmaceutical products, food and beverage, and feeds, or may beformulated into powders, granules, tablets, capsules, drinks and anyother preparation in accordance with conventional methods. Thesensation-improving agent according to the present invention can be usedas application agent in any conventional application form, such asemulsion, cream, lotion, or pack. These application agents may beproduced through conventional methods by appropriately adding amilk-derived protein and/or a hydrolysate therefrom as an activeingredient in the present invention in the course of production, and canalso be used as cosmetics. Also, other components that have asensation-improving effect, for example, ceramide, sphingomyelinase, andsphingomyelin can be used in combination with the milk-derived proteinand/or the hydrolysate therefrom. In the test described below for theeffective amount of the sensation-improving agent according to thepresent invention, the milk-derived protein and/or the hydrolysatetherefrom was orally ingested in a mouse at a dose of 10 mg or more,preferably 20 mg or more per kg of body weight of the mouse to improvethe peripheral sensations in the mouse. The dulling of sensations,particularly peripheral sensations can be expected to be improved byingesting the milk-derived protein and/or the hydrolysate therefromtypically at a dose of 10 mg or more, preferably 20 mg or more per dayfor an adult human. It is desirable to ensure the ingestion at thisnecessary dose. If applied as an application agent to the skin, thedensity of the applied milk-derived protein and/or hydrolysate therefromis 0.001 to 40% by weight, more preferably 0.1 to 10% by weight based onthe total weight of the application agent.

The sensation-improving food, beverage according to the presentinvention may be produced by adding the milk-derived protein and/or thehydrolysate therefrom with a conventional food, beverage, for example,yoghurt, a milk beverage, a wafer, and a dessert. Depending on the formof these sensation-improving food, beverage, a milk-derived proteinand/or a hydrolysate therefrom is preferably combined in an amount of0.5 to 2000 mg per 100 g of the food, beverage in order to take a humanthe milk-derived protein and/or the hydrolysate therefrom at a dose of10 mg or more per day for an adult human. The sensation-improving feedaccording to the present invention may be produced by adding themilk-derived protein and/or the hydrolysate therefrom with aconventional feed, for example, a feed for domestic animals and a petfood. For example, if these feeds contain the milk-derived proteinand/or the hydrolysate therefrom, a milk-derived protein and/or ahydrolysate therefrom is preferably added in an amount of 0.5 to 2000 mgper 100 g of the feed in order to feed a mammal the milk-derived proteinand/or the hydrolysate therefrom at a dose of 10 mg or more.

The milk-derived protein and/or the hydrolysate therefrom may becombined by any method in the present invention. For example, foraddition in solution, a milk-derived protein and/or a hydrolysatetherefrom is suspended or dissolved in deionized water, and the mixtureis stirred followed by formulation of the mixture into the form of apharmaceutical product, a food, beverage, and a feed. The milk-derivedprotein and/or the hydrolysate therefrom in deionized water is stirredunder such conditions that the milk-derived protein and/or a hydrolysatetherefrom may be homogeneously mixed, and can be mixed with anultradisperser or a TK homomixer. The solution can be concentrated withan RO membrane or freeze-dried, if necessary, to be readily used for apharmaceutical product, a food, beverage, and a feed. The formulationprocess in the present invention can include sterilization treatmentcommonly used in the manufacture of pharmaceutical products, food,beverage, and feeds, and for the formulation in the form of powder,dry-heat sterilization. Accordingly, a pharmaceutical product, a food,beverage, and a feed that contain a milk-derived protein and/or ahydrolysate therefrom according to the present invention can be producedin various forms such as liquid, gel, powder, or granule form.

The present invention will be described below in detail by way ofexamples and test examples, which are illustrative only and not intendedto be limiting the present invention in any way.

Example 1

A column (diameter 5 cm and height 30 cm) filled with 400 g of a cationexchange resin, sulfonated Chitopearl (from Fuji Spinning Co., Ltd.) wasthoroughly washed with deionized water, and 40 L of unsterilized skimmilk (pH 6.7) was then passed through the column at a flow rate of 25ml/min. The column was then thoroughly washed with deionized water, andthe adsorbate was eluted with a 0.02 M carbonate buffer solutioncontaining 2.0 M sodium chloride (pH 7.0). Eluted fractions containinglactoperoxidase were allowed to apply on an S-Sepharose FF column (fromGE Healthcare Ltd.), and the column was thoroughly washed with deionizedwater, and was equilibrated with a 10 mM phosphate buffer solution (pH7.0). The adsorbate was then eluted by a linear gradient of 0-2.0 Msodium chloride to collect a fraction containing lactoperoxidase. Thefraction was treated by gel filtration chromatography on HiLoad 16/60Superdex 75 pg (from GE Healthcare Ltd.) to yield 3.0 g oflactoperoxidase. The resulting lactoperoxidase had a purity of 94%, andcan be used as a sensation-improving agent (Example Product 1) withoutfurther purification.

Example 2

Lactoperoxidase (1 g) prepared in Example 1 was dissolved in 200 ml ofwater, and a pancreatin (from Sigma Co.) was added to the solution intoa final concentration of 0.01% by weight. The solution was treated withthe enzyme at 37° C. for 5 hours. The mixture was heat-treated at 90° C.for 5 minutes to deactivate the enzyme, and was freeze-dried to yield0.8 g of a hydrolysate from lactoperoxidase. The resulting hydrolysatefrom lactoperoxidase had a molecular weight of 10,000 or less, and canbe used as a sensation-improving agent (Example Product 2) withoutfurther purification.

Example 3

Lactoperoxidase (1 g) prepared in Example 1 was dissolved in 200 ml ofwater, and a trypsin (from Sigma Co.) was added to the solution into afinal concentration of 0.01% by weight. The solution was treated withthe enzyme at 37° C. for 5 hours. The mixture was heat-treated at 90° C.for 5 minutes to deactivate the enzyme, and was freeze-dried to yield0.9 g of a hydrolysate from lactoperoxidase. The resulting hydrolysatefrom lactoperoxidase had a molecular weight of 10,000 or less, and canbe used as a sensation-improving agent (Example Product 3) withoutfurther purification.

Example 4

A column (diameter 5 cm and height 30 cm) filled with 400 g of a cationexchange resin sulfonated Chitopearl (from Fuji Spinning Co., Ltd.) wasthoroughly washed with deionized water, and 40 L of unsterilized skimmilk (pH 6.7) was then passed through the column at a flow rate of 25ml/min. The column was then thoroughly washed with deionized water, andthe adsorbate was eluted with a 0.02 M carbonate buffer solutioncontaining 2.0 M sodium chloride (pH 7.0). Eluted fractions containinglactoferrin were allowed to apply on an S-Sepharose FF column (from GEHealthcare Ltd.), and the column was thoroughly washed with deionizedwater, and was equilibrated with a 10 mM phosphate buffer solution (pH7.0). The adsorbate was then eluted by a linear gradient of 0-2.0 Msodium chloride to collect a fraction containing lactoferrin. Thefraction was treated by gel filtration chromatography on HiLoad 16/60Superdex 75 pg (from GE Healthcare Ltd.) to yield 8.0 g of lactoferrin.The resulting lactoferrin had a purity of 96%, and can be used as asensation-improving agent (Example Product 4) without furtherpurification.

Example 5

Lactoferrin (1 g) prepared in Example 4 was dissolved in 200 ml ofwater, and a pancreatin (from Sigma Co.) was added to the solution intoa final concentration of 0.01% by weight. The solution was treated withthe protease at 37° C. for 5 hours. The mixture was heat-treated at 90°C. for 5 minutes to deactivate the enzyme, and was freeze-dried to yield0.8 g of a hydrolysate from lactoferrin. The resulting hydrolysate fromlactoferrin had a molecular weight of 10,000 or less, and can be used asa sensation-improving agent (Example Product 5) without furtherpurification.

Example 6

Lactoferrin (1 g) prepared in Example 4 was dissolved in 200 ml ofwater, and a trypsin (from Sigma Co.) was added to the solution into afinal concentration of 0.01% by weight. The solution was treated withthe protease at 37° C. for 5 hours. The mixture was heat-treated at 90°C. for 5 minutes to deactivate the enzyme, and was freeze-dried to yield0.9 g of a hydrolysate from lactoferrin. The resulting hydrolysate fromlactoferrin had a molecular weight of 10,000 or less, and can be used asa sensation-improving agent (Example Product 6) without furtherpurification.

Example 7

A column filled with 3,000 g of S-Sepharose was thoroughly washed withdeionized water, and 10,000 L of skim milk was then passed through thecolumn. The column was thoroughly washed with deionized water, and theadsorbate was then eluted by a linear concentration gradient with 0.1 to1.0 M sodium chloride. The resulting fractions were heat-treated at 90°C. for 10 minutes and were then centrifuged to remove precipitates. Theeluted fraction containing bovine milk-derived basic cystatin was againfractionated by Mono S ion exchange chromatography. This fraction wastreated sequentially by Mono Q ion exchange chromatography and Superose12 gel filtration chromatography in an FPLC system, and subsequentlyhydroxyapatite chromatography and C4 reverse phase chromatography in anHPLC system to yield 58 mg of cystatin (Fraction A). The resultingcystatin can be used as a sensation-improving agent (Example Product 7)without further purification.

Example 8

A 5% whey protein solution (10,000 L) was heat-treated at 90° C. for 10minutes, and was then centrifuged to remove precipitates. A column wasfilled with a carrier prepared by binding carboxymethylated papain toTresyl-Toyopearl (from TOSOH CORPORATION), and was then equilibratedwith a 0.5 M sodium chloride solution. The above-described whey proteinsolution was passed through the column. The column was washed with a 0.5M sodium chloride solution and then a 0.5 M sodium chloride solutioncontaining 0.1% Tween 20. Cysteine protease was then eluted with a 20 mMacetic acid-0.5 M sodium chloride solution. Eluted fractions wereimmediately neutralized with a 1 M sodium hydroxide solution. Theneutralized solution was fractionated by Mono S anion exchangechromatography, hydroxyapatite chromatography, and then C4 reverse phasechromatography in an HPLC system to yield 48 mg of milk-derived basiccystatin (Fraction B). The resulting cystatin can be used as asensation-improving agent (Example Product 8) without furtherpurification.

Example 9

Fraction A (25 mg) prepared in Example 7 was suspended in 100 ml ofwater, and pancreatin was added to the suspension into a finalconcentration of 1% by weight. The suspension was treated with theenzyme at 37° C. for 5 hours. The suspension was heat-treated at 90° C.for 5 minutes to deactivate the enzyme, and was freeze-dried to yield 23mg of a hydrolysate from cystatin (Fraction C). The fraction B (25 mg)prepared in Example 8 was treated in a similar manner to yield 24 mg ofa hydrolysate from cystatin (Fraction D). The resulting hydrolysate fromcystatin had a molecular weight of 8,000 or less, and can be used as asensation-improving agent (Example Product 9) without furtherpurification.

Example 10

A column (diameter 5 cm and height 30 cm) filled with 400 g of a cationexchange resin sulfonated Chitopearl (from Fuji Spinning Co., Ltd.) wasthoroughly washed with deionized water, and 40 L of unsterilized skimmilk (pH 6.7) was then passed through the column at a flow rate of 25ml/min. The column was then thoroughly washed with deionized water, andthe adsorbate was eluted with a 0.02 M carbonate buffer solutioncontaining 2.0 M sodium chloride (pH 7.0). Eluted fractions containingangiogenin were allowed to apply on an S-Sepharose FF column (from GEHealthcare Ltd.), and the column was thoroughly washed with deionizedwater, and was equilibrated with a 10 mM phosphate buffer solution (pH7.0). The adsorbate was then eluted by a linear gradient of 0-2.0 Msodium chloride to collect a fraction containing angiogenin. Thefraction was treated by gel filtration chromatography on HiLoad 16/60Superdex 75 pg (from GE Healthcare Ltd.) to yield 1.8 g of a fractionabundantly containing angiogenin. The angiogenin content in theresulting fraction abundantly containing angiogenin is 10%, and thefraction can be used as a sensation-improving agent (Example Product10).

Example 11

A column filled with 3,000 g of a cation exchange resin, sulfonatedChitopearl (from Fuji Spinning Co., Ltd.) was thoroughly washed withdeionized water, and 100 L of unsterilized skim milk (pH 6.7) was thenpassed through the column. This column was then thoroughly washed withdeionized water, and the adsorbate was eluted by a linear concentrationgradient of 0.1 to 2.0 M sodium chloride. The eluted fraction containingangiogenin was fractionated by S-Sepharose cation exchangechromatography (from GE Healthcare Ltd.), the resulting fractioncontaining angiogenin was heat-treated at 90° C. for 10 minutes, and wascentrifuged to remove precipitates. This fraction containing angiogeninwas sequentially treated by Mono S cation exchange chromatography,Superose 12 gel filtration chromatography, hydroxyapatitechromatography, and C4 reverse phase chromatography to yield 55 mg ofangiogenin. The resulting angiogenin had a purity of 99%, and can beused as a sensation-improving agent (Example Product 11).

Example 12

Angiogenin (5 mg) prepared in Example 11 was dissolved in 10 ml ofwater, and pancreatin (from Sigma Co.) was added to the solution into afinal concentration of 0.01% by weight. The solution was treated withthe enzyme at 37° C. for 5 hours. The solution was heat-treated at 90°C. for 5 minutes to deactivate the enzyme, and was freeze-dried to yield4.0 mg of a hydrolysate from angiogenin. The resulting hydrolysate fromangiogenin had a molecular weight of 10,000 or less, and can be used asa sensation-improving agent (Example Product 12) without furtherpurification.

Example 13

Angiogenin (5 mg) prepared in Example 11 was dissolved in 10 ml ofwater, and trypsin (from Sigma Co.) was added to the solution into afinal concentration of 0.01% by weight. The solution was treated withthe enzyme at 37° C. for 5 hours. The solution was heat-treated at 90°C. for 5 minutes to deactivate the enzyme, and was freeze-dried to yield4.2 mg of a hydrolysate from angiogenin. The resulting hydrolysate fromangiogenin had a molecular weight of 10,000 or less, and can be used asa sensation-improving agent (Example Product 13) without furtherpurification.

Test Example 1 Verification of Promotion of Cell Differentiation

3T3 cells being a fibroblast cell line that is known to be present inthe skin were incubated for two days in the presence of Example Products1, 3, 4, 5, and 7, Fraction C in Example Product 9, and Example Products11 and 13, each in a concentration of 0.03 to 1%. As a control, 3T3cells were incubated for two days in the absence of any Example Product(control). PC-12 cells being neural model cells were incubated withthose culture supernatants, and morphological differentiation of thePC-12 cells was observed when a neurotrophic factor was secreted by 3T3cells.

The results showed that all of the culture supernatants containing anExample Product differentiated PC-12 cells clearly. This experiment wasrepeated several times, and the proportion of differentiation wasobserved with an optical microscope. When any Example Product was added,differentiation was observed in 95% or more of the cells. In contrast,the control culture supernatant failed to differentiate PC-12 cells, andobservation with an optical microscope in the experiment that wasrepeated several times revealed no differentiation. This shows that amilk-derived protein and/or a hydrolysate therefrom promotes thesecretion of a neurotrophic factor from 3T3 cells, and promotes thedifferentiation of PC-12 cells being neural model cells.

Test Example 2 Verification of Sensation-Improving Effect in AnimalExperiments

A sensation-improving effect by thermal stimulation was evaluated in thehot plate that is a behavioral study to thermal stimulation developed byWoolfe, MacDonald, et al. 24-weeks old hairless mice (Hos:HR-1) weredivided into 13 groups with 6 mice in each group. Example Products 2, 6,and 8, Fraction D in Example Product 9, Example Products 10 and 12 wereeach orally administered to a mouse through a sonde in a dose of 10 mgor 20 mg once daily per kg body weight in a mouse, or a vehicle only wasorally administered to a mouse through a tube once daily (control; 0mg), and these mice were bred for 4 weeks. At the end of theadministration, mice were placed on a hot plate at 54° C., and the timeuntil the mice exhibited escape behavior, such as pulling their pawsaway from the hot plate, standing up, and jumping was measured. Themaximal strength of thermal stimulation was set to 30 seconds, the valueat this maximal strength was assigned to 30 seconds. The results areshown in Table 1.

TABLE 1 escape behavior positive response time control  0 mg 29.2 ± 0.14seconds Example Product 2 10 mg 22.2 ± 0.17 seconds 20 mg 20.1 ± 0.25seconds Example Product 6 10 mg 22.8 ± 0.18 seconds 20 mg 20.5 ± 0.22seconds Example Product 8 10 mg 23.1 ± 0.27 seconds 20 mg 21.0 ± 0.13seconds Example Product 9 10 mg 23.3 ± 0.10 seconds (Fraction D) 20 mg21.1 ± 0.21 seconds Example Product 10 10 mg 26.2 ± 0.17 seconds 20 mg24.1 ± 0.16 seconds Example Product 12 10 mg 23.9 ± 0.21 seconds 20 mg21.8 ± 0.20 seconds

Table 1 demonstrates that ingestion of Example Products 2, 6, and 8,Fraction D in Example Product 9, and Example Products 10 and 12 eachshow a tendency toward a shorter escape behavior positive response timeat a dose of 10 mg, and significantly shortened the time at a dose of 20mg. This indicates that ingestion of Example Products 2, 6, and 8,Fraction D in Example Product 9, and Example Products 10 and 12 canprevent or improve the dulling of sensations, particularly peripheralsensations.

Test Example 3 Verification of Sensation-Improving Effect by OralIngestion

Healthy elderly subjects (average age 75±3) who experienced dulledsensations in the hand were divided into 9 groups with 10 subjects ineach group. These groups consisted of Group A with ingestion of noExample Product, Group B with ingestion of Example Product 1 at a doseof 10 mg, Group C with ingestion of Example Product 1 at a dose of 20mg, Group D with ingestion of Example Product 4 at a dose of 10 mg,Group E with ingestion of Example Product 4 at a dose of 20 mg, Group Fwith ingestion of Example Product 7 at a dose of 10 mg, Group G withingestion of Example Product 7 at a dose of 20 mg, Group H withingestion of Example Product 11 at a dose of 10 mg, and Group I withingestion of Example Product 11 at a dose of 20 mg, and such ingestionwas continued for 6 weeks. As determined with an algesiometer (fromIntercross) which is an instrument for determining superficialsensations in accordance with the manufacturer's directions for usebefore and after the 6-week ingestion, pain sensations in the palm ofthe hand and the sole of the foot were graded in four ranks from normalto declines I to III on the basis of pain sensations in the medial sideof the arm. The results are shown in Tables 2 and 3. Moreover, after the6-week ingestion, a questionnaire survey was conducted to each subjecton the improvement of his/her sensation in the hand. The results areshown in Tables 4 and 5.

(Measurement)

The pain sensation was evaluated using five pins that have differentthicknesses in combination with five positions of a fulcrum. Thethinnest pin 1 was rolled along the medial side of the arm, and thesubject was asked about the degree of normal pain sensation. The pin 1was then rolled along the palm and the sole of the foot while theposition of the fulcrum for the holder was sequentially changed todetermine the position of the fulcrum at which the same degree of painsensation as the first pain sensation was caused.

(Evaluation)

The algesiometer was designed to cause pain sensations in the samedegree in rolling the pin 1 (fulcrum: 50 g) along the medial side of thearm and in rolling the pin 2 (fulcrum: 50 g) along the palm, and wasused in accordance with the manufacturer's directions for use toevaluate the pain sensation as described below. The evaluation of thepain was scored and the scores were averaged.

Normal (score 0): The pain sensation in the same degree was caused inrolling the pin 2 (50 g)

Decline I (score 1): The pain sensation in the same degree was caused inrolling the pin 1 (50 g)

Decline II (score 2): The pain sensation in the same degree was causedin rolling the pin 1 (60 g)

Decline III (score 3): The pain sensation in the same degree was causedin rolling the pin 1 (70 g)

Decline Decline Decline Average Normal I II III value Measurement ofsensation in the hand (Before ingestion) Group A 0 2 3 5 2.3 Group B 0 15 4 2.3 Group C 0 1 5 4 2.3 Group D 0 1 4 5 2.4 Group E 0 1 5 4 2.3Group F 0 2 3 5 2.3 Group G 0 1 5 4 2.3 Group H 0 1 5 4 2.3 Group I 0 14 5 2.4 Measurement of sensation in the hand (After 6-week ingestion)Group A 0 2 4 4 2.2 Group B 0 3 5 2 1.9 Group C 2 4 3 1 1.3 Group D 1 24 3 1.9 Group E 2 2 5 1 1.5 Group F 0 3 5 2 1.9 Group G 2 4 3 1 1.3Group H 1 2 4 3 1.9 Group I 2 2 5 1 1.5

TABLE 3 Decline Decline Decline Average Normal I II III valueMeasurement of sensation in the sole of the foot (Before ingestion)Group A 0 2 3 5 2.3 Group B 0 1 4 5 2.4 Group C 0 1 3 6 2.5 Group D 0 16 3 2.2 Group E 0 1 4 5 2.4 Group F 0 2 3 5 2.3 Group G 0 1 4 5 2.4Group H 0 1 3 6 2.5 Group I 0 1 6 3 2.2 Measurement of sensation in thesole of the foot (After 6-week ingestion) Group A 0 2 3 5 2.3 Group B 14 1 4 1.8 Group C 2 3 3 2 1.5 Group D 0 3 6 1 1.8 Group E 1 3 5 1 1.6Group F 1 4 1 4 1.8 Group G 2 3 3 2 1.5 Group H 0 3 6 1 1.8 Group I 1 35 1 1.6

TABLE 4 Sensation in the hand Deteriorated Unchanged Recovered Group A 27 1 Group B 1 5 4 Group C 0 2 8 Group D 0 3 7 Group E 0 2 8 Group F 1 54 Group G 0 2 8 Group H 0 3 7 Group I 0 2 8

TABLE 5 Sensation in the sole of the foot Deteriorated UnchangedRecovered Group A 2 7 1 Group B 0 6 4 Group C 0 2 8 Group D 0 5 5 GroupE 0 2 8 Group F 0 6 4 Group G 0 2 8 Group H 0 5 5 Group I 0 2 8

Tables 2 to 5 demonstrate that ingestion of Example Products 1, 4, 7,and Example Product 11 each show a tendency toward improved sensationsin the hand and the sole of the foot at a dose of 10 mg, andsignificantly improved the sensations at a dose of 20 mg. The dulledsensations, particularly peripheral sensations can be expected to beimproved by ingesting the milk-derived protein and/or the hydrolysatetherefrom typically at a dose of 10 mg or more, preferably 20 mg or moreper day for an adult human.

Example 14 Preparation of Sensation-Improving Cosmetic Product (Cream)

A hydrolysate from lactoperoxidase prepared in Example 3 (ExampleProduct 3) was used to produce a sensation-improving cosmetic product(cream) by mixing with raw materials in the proportion shown in Table 6.

TABLE 6 Line 6: Hydrolysate from lactoperoxidase Glyceryl monostearate(self-emulsifiable) 10.0 Purified lanolin 6.0 Liquid paraffin 5.0 Jojobaoil 5.0 Paraben 0.3 Decomposed product from lactoperoxidase 1.0 (ExampleProduct 3) Flavoring agent Appropriate amount Sterile ion exchangedwater q.s to 100.0

Test Example 4 Test for Sensation-Improving Effect by Application to theSkin

Healthy elderly subjects (average age 75±3) who experienced dulledsensations in the hand were divided into 2 groups, Group A and Group Bwith 15 subjects in each group. A cosmetic product (cream) prepared asin Example Product 14 except that any sensation-improving agent was notcontained was applied to subjects in Group A, and a sensation-improvingcosmetic product (cream) in Example Product 14 to subjects in Group Bonce daily over their hands and feet. The application was continued for6 weeks. As determined with an algesiometer (from Intercross) which isan instrument for determining superficial sensations in accordance withthe manufacturer's directions for use before and after the 6-weekapplication, pain sensations in the palm of the hand and the sole of thefoot were graded in four ranks from normal to declines I to III on thebasis of pain sensations in the medial side of the arm. The results areshown in Tables 7 and 8. Moreover, after the 6-week application, aquestionnaire survey was conducted to each subject on the improvement ofhis/her sensation in the hand. The results are shown in Tables 9 and 10.The measurement was carried out as in Test example 3.

TABLE 7 Decline Decline Decline Average Normal I II III valueMeasurement of sensation in the hand (Before application) Group A 0 4 65 2.1 Group B 0 5 4 6 2.1 Measurement of sensation in the hand (After6-week application) Group A 1 3 7 4 1.9 Group B 3 6 3 3 1.4

TABLE 8 Decline Decline Decline Average Normal I II III valueMeasurement of sensation in the sole of the foot (Before application)Group A 0 5 5 5 2.0 Group B 0 5 6 4 1.9 Measurement of sensation in thesole of the foot (After 6-week application) Group A 1 3 5 6 2.1 Group B2 6 6 1 1.4

TABLE 9 Sensation in the hand Deteriorated Unchanged Recovered Group A 212 1 Group B 1 6 8

TABLE 10 Sensation in the sole of the foot Deteriorated UnchangedRecovered Group A 1 13 1 Group B 0 9 6

Tables 7 to 10 demonstrate that application of the sensation-improvingcosmetic product (cream) in Example Product 14 shows a tendency towardimproved sensations in the hand and the sole of the foot. This indicatesthat the dulled peripheral sensations can be expected to be improved byapplying the cream containing a sensation-improving agent according tothe present invention.

Example 15 Preparation of Sensation-Improving Liquid NutrientComposition

A hydrolysate (5 g) from lactoferrin in Example Product 5 was dissolvedin 4995 g of deionized water, the solution was stirred with a TKhomomixer (TKROBO MICS; from Tokusyukika) at 6000 rpm for 30 minutes toprepare a solution of the hydrolysate from lactoferrin having a contentof the hydrolysate from lactoferrin of 100 mg/100 g. To 5.0 kg of thesolution of the hydrolysate from lactoferrin were added 4.0 kg ofcasein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perillaoil, 18.0 kg of dextrin, 6.0 kg of a mineral mixture, 1.95 kg of avitamin mixture, an 2.0 kg of emulsifier, 4.0 kg of stabilizer, and 0.05kg of a flavoring agent. The mixture was then placed into a 200 mlretort pouch. The retort pouch was sterilized with a retort sterilizer(Class-1 pressure vessel, TYPE: RCS-4CRTGN, from HISAKA WORKS, LTD.) at121° C. for 20 minutes to prepare 50 kg of a sensation-improving liquidnutrient composition according to the present invention. The resultingsensation-improving liquid nutrient composition had no precipitate, norany abnormality of taste. The sensation-improving liquid nutrientcomposition had a content of the hydrolysate from lactoferrin of 10mg/100 g.

Example 16 Preparation of Sensation-Improving Gel Food

Cystatin (2 g) in Example Product 8 was dissolved in 708 g of deionizedwater, and the solution was stirred with an ultra-disperser(ULTRA-TURRAXT-25; from IKA Japan) at 9500 rpm for 30 minutes. To thesolution were added 40 g of sorbitol, 2 g of an acidulant, 2 g of aflavoring agent, 5 g of pectin, 5 g of a whey protein concentrate, 1 gof calcium lactate, and 235 g of deionized water. The mixture wasstirred and was placed into a 200 ml cheer pack. The cheer pack wassterilized at 85° C. for 20 minutes, and was then sealed to prepare 5bags (each 200 g) of a sensation-improving gel food according to thepresent invention. The resulting sensation-improving gel food had noprecipitate, nor any abnormality of taste. The sensation-improving gelfood had a cystatin content of 200 mg/100 g.

Example 17 Preparation of Sensation-Improving Beverage

An acidulant (2 g) was dissolved in 706 g of deionized water, and 4 g ofangiogenin in Example Product 11 was then dissolved in the solution. Themixture was stirred with an ultra-disperser (ULTRA-TURRAXT-25; from IKAJapan) at 9500 rpm for 30 minutes. To the mixture were added 100 g ofmaltitol, 20 g of a reduced starch syrup, 2 g of a flavoring agent, and166 g of deionized water, and the mixture was filled into a 100 ml glassbottle. The bottle was sterilized at 95° C. for 15 seconds and wassealed to prepare 10 bottles (each 100 ml) of a sensation-improvingbeverage. The resulting sensation-improving beverage had no precipitate,nor any abnormality of taste. The sensation-improving beverage had anangiogenin content of 400 mg/100 g.

Example 18 Preparation of Sensation-Improving Feed

A hydrolysate from lactoperoxidase (2 kg) in Example Product 2 wasdissolved in 98 kg of deionized water, and the solution was stirred witha TK homomixer (MARK II Model 160; from Tokusyukika) at 3600 rpm for 40minutes to prepare a solution of a hydrolysate from lactoperoxidasehaving a content of the hydrolysate from lactoperoxidase of 2 g/100 g.To 10 kg of the solution of the hydrolysate from lactoperoxidase wereadded 12 kg of soybean cake, 14 kg of skim milk powder, 4 kg of soybeanoil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kgof wheat flour, 2 kg of bran, 5 kg of a vitamin mixture, 2.8 kg ofcellulose, and 2 kg of a mineral mixture, and the mixture was sterilizedat 120° C. for 4 minutes to prepare 100 kg of a sensation-improvingcanine feed according to the present invention. The sensation-improvingcanine feed had a content of the hydrolysate from lactoperoxidase of 200mg/100 g.

Example 19 Preparation of Sensation-Improving Agent (Tablet)

The raw materials were mixed in the proportion shown in Table 11, andthe mixture was shaped into 1-g tablets in accordance with aconventional method to prepare a sensation-improving agent according tothe present invention. The sensation-improving agent had alactoperoxidase content of 100 mg/g.

TABLE 11 Hydrous crystalline glucose 83.5% (% by weight) Lactoperoxidase(Example Product 1) 10.0% Mineral mixture 5.0% Sugar ester 1.0%Flavoring agent 0.5%

Example 20 Preparation of Sensation-Improving Cosmetic Product (Lotion)

The raw materials were mixed in the proportion shown in Table 12 toprepare a sensation-improving cosmetic product (lotion).

TABLE 12 Sorbitol 3.0 Sodium DL-pyrrolidone carboxylate 2.0Carboxymethyl cellulose 0.3 Paraben 0.1 Lactoferrin (Example Product 4)1.5 Flavoring agent Appropriate amount Sterile ion exchanged water q.sto 100.0

1. A sensation-improving agent containing a milk-derived protein and/ora hydrolysate therefrom as an active ingredient.
 2. Thesensation-improving agent according to claim 1, wherein the milk-derivedprotein is at least one selected from lactoperoxidase, lactoferrin,cystatin, and angiogenin.
 3. The sensation-improving agent according toclaim 1, wherein the hydrolysate from the milk-derived protein isproduced through hydrolisis of the milk-derived protein with a protease.4. The sensation-improving agent according to claim 3, wherein theprotease is at least one selected from the group consisting of pepsin,trypsin, chymotrypsin, and pancreatin.
 5. A sensation-improving food,beverage, feed, or cosmetics containing the component according toclaim
 1. 6. A method for improving a sensation in a mammal, comprisingtaking the mammal a milk-derived protein and/or a hydrolysate therefrom,or applying the milk-derived protein and/or the hydrolysate therefrom tothe skin of the mammal.
 7. The method according to claim 6, wherein themammal is a human, and the milk-derived protein and/or the hydrolysatetherefrom is fed at a dose of 10 mg or more per day for an adult human.8. The sensation-improving agent according to claim 2, wherein thehydrolysate from the milk-derived protein is produced through hydrolisisof the milk-derived protein with a protease.
 9. The sensation-improvingagent according to claim 8, wherein the protease is at least oneselected from the group consisting of pepsin, trypsin, chymotrypsin, andpancreatin.
 10. A sensation-improving food, beverage, feed, or cosmeticscontaining the component according to claim
 2. 11. A sensation-improvingfood, beverage, feed, or cosmetics containing the component according toclaim
 3. 12. A sensation-improving food, beverage, feed, or cosmeticscontaining the component according to claim
 4. 13. A sensation-improvingfood, beverage, feed, or cosmetics containing the component according toclaim
 8. 14. A sensation-improving food, beverage, feed, or cosmeticscontaining the component according to claim 9.